Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

Kindle, Karen L.; Lawrence, Susan D.

doi:10.1104/pp.116.3.1179

Cited by 24 publications

(9 citation statements)

References 59 publications

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“…We hypothesized that some cytosolic factors (absents in the in vitro import assays) could play a role in the (⌬1-59)-ceQORH::GFP plastid targeting in planta. In this way, similar differences between in vitro experiments and in planta observations were also described by other authors and indicate that in vitro import assays may not fully match the more complex in vivo situation suggesting that some cytosolic components provide back-up systems to assure import of certain precursors under different environmental conditions (64,65).…”

Section: In Vitro and In Planta Experiments Indicate That The C Termisupporting

confidence: 81%

Toc159- and Toc75-independent Import of a Transit Sequence-less Precursor into the Inner Envelope of Chloroplasts

Miras

Salvi

Piette

et al. 2007

Journal of Biological Chemistry

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Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.Plastids conduct vital biosynthetic functions, and many essential reactions are located exclusively within these organelles. However, plastids contain only limited coding information in their own DNA. Endosymbiotic evolution has resulted in the transfer to the nuclear genome of genes encoding the vast majority of plastid proteins. As a consequence of this displacement of genetic material, plastids had to evolve mechanisms to reimport cytoplasmically synthesized precursor proteins from the cytosol (1-6). In most cases, nucleus-encoded plastid proteins are synthesized in the cytosol as higher molecular weight precursors, with a cleavable N-terminal sequence called transit peptide (7). Transit peptides contain all the information that is necessary and sufficient for import. During the actual import process, transit peptides are proteolytically removed by a stromal processing peptidase. Transit peptides can be simple, as found for stroma proteins, or bipartite, as found for proteins destined to thylakoids. In the latter case, the N-terminal part directs the precursor to the stroma, whereas the non-cleaved C-terminal part directs the partially processed precursors to their final intraorganellar destination; i.e. the thylakoid membranes and the thylakoid lumen. Ultimate precursor maturation occurs by virtue of the thylakoid-processing peptidase (8).During chloroplast import, the transit peptide first recognizes the chloroplast surface in a process involving membrane lipids and the translocon at the outer chloroplast envelope (TOC) 3 (9). The TOC complex consists of three distinct core subunits: the GTP-dependent Toc34 and Toc159 receptors and the translocation channel protein Toc75 (10, 11). Translocation across the inner envelope is mediated by another multiprotein complex, the translocon at the inner chloroplast envelope (TIC) and requires ATP in the st...

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Section: In Vitro and In Planta Experiments Indicate That The C Termisupporting

confidence: 81%

Toc159- and Toc75-independent Import of a Transit Sequence-less Precursor into the Inner Envelope of Chloroplasts

Miras

Salvi

Piette

et al. 2007

Journal of Biological Chemistry

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“…Regardless of the molecular mechanism, however, our findings clearly suggest that accessibility of the N terminus of SS-tp is required for interaction with one or more components of the chloroplast translocation apparatus. Several investigations into the role of different domains of chloroplast transit peptides have also shown that the N terminus is required for precursor import in vitro (24,25), in vivo (26,27), and for directing the import of certain fusion proteins in vitro (8). An intriguing aspect of these reports is that N terminus of some transit peptides is often the least conserved region of the entire presequence (26).…”

Section: Discussionmentioning

confidence: 99%

The C Terminus of a Chloroplast Precursor Modulates Its Interaction with the Translocation Apparatus and PIRAC

Dabney‐Smith

Wijngaard

Treece

et al. 1999

Journal of Biological Chemistry

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The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (⌬52, ⌬67, and ⌬74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of 35 S-prSSU. Both IC 50 and K i values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and ⌬52 were properly processed and accumulated within the chloroplast, whereas ⌬67 and ⌬74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.

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“…(29, 30, 99) Site-directed mutations in nuclear AtpC gene (114,199) Induced mutations in chloroplast atpE, atpF and atpI genes See (44) Nuclear mutation affecting expression of the chloroplast-encoded atpH and atpI genes…”

Section: Photophosphorylationmentioning

confidence: 99%

CHLAMYDOMONAS AS A MODEL ORGANISM

Harris

2001

Annu. Rev. Plant. Physiol. Plant. Mol. Biol.

612

447

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The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

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Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

Cited by 24 publications

References 59 publications

Toc159- and Toc75-independent Import of a Transit Sequence-less Precursor into the Inner Envelope of Chloroplasts

Toc159- and Toc75-independent Import of a Transit Sequence-less Precursor into the Inner Envelope of Chloroplasts

The C Terminus of a Chloroplast Precursor Modulates Its Interaction with the Translocation Apparatus and PIRAC

CHLAMYDOMONAS AS A MODEL ORGANISM

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